Roles of intracellular calcium and NF-kappa B in the Clostridium difficiletoxin A-induced up-regulation and secretion of IL-8 from human monocytes

Clostridium difficile causes an intense inflammatory colitis through the ac tions of two large exotoxins, toxin A and toxin B. IL-8 is believed to play an important role in the pathophysiology of C. difficile-mediated colitis, although the mechanism whereby the toxins up-regulate the release of IL-8 from target cells is not wed understood. In this study, we investigated the mechanisms through which toxin A induces IL-8 secretion in human monocytes , We found that cellular uptake of toxin A is required for the up-regulatio n of IL-8, an effect that is not duplicated by a recombinant toxin fragment comprising the cell-binding domain alone, Toxin A induced IL-8 expression at the level of gene transcription and this effect occurred through a mecha nism requiring intracellular calcium and calmodulin activation. Additionall y, the effects of toxin A were inhibited by the protein tyrosine kinase inh ibitor genistein, but were unaffected by inhibitors of protein kinase C and phosphatidylinositol-3 kinase. We determined that toxin A activates nuclea r translocation of the transcription factors NF-kappa B and AP-1, but not N F-IL-6. NF-kappa B inhibitors blocked the ability of toxin A to induce IL-8 secretion, and supershift analysis indicated that the major isoform of NF- kappa B activated by the toxin is a p50-p65 heterodimer. This study is the first to identify intracellular signaling pathways and transcription factor s involved in the C. difficile toxin-mediated up-regulation of IL-8 synthes is and release by target cells. This information should increase our unders tanding of the pathogenesis of C. difficile colitis and the nature of IL-8 gene regulation as well.